Project Summary Stable genetic modification of autologous stem cells using HIV-1 derived lentivirus vectors confers clinical benefit in patients with inherited disorders of hematopoiesis and immunity. Broad tropism for gene delivery to diverse tissues, stable passage of the proviral integrant to all progeny, and the ability to scale production for clinical use provide strong motivation for using this vector system in stem cell gene therapy. Nevertheless, instances of insertional activation of endogenous genes continue to raise concerns for the use of integrating lentivectors in hematopoietic stem cell gene therapy. Current non- integrating vectors provide long-term expression in postmitotic tissues, but failure to replicate the episomal vector genome leads to rapid loss in dividing stem cell populations. We recently reported the development of a novel, non-integrating episomal lentivector that faithfully replicates through cell division and expresses a gene of interest - without selection. In distinction to similar vectors, this anchored Non-Integrating LentiVector (aNILV) avoids heterologous viral sequences and relies on human b-interferon nuclear scaffold/matrix attachment region (S/MAR) locus sequences. Our principal hypothesis is that aNILV will optimize biosafety and provide long-term correction of bone marrow failure in a murine model of Fanconi Anemia. We will develop a second-generation episome vector design (aNILV-II) additionally capable of conversion to backbone-free minicircles to further enhance biosafety and long-term transgene expression. This proposal is uniquely responsive to PAS-13-006: New Directions in Hematology Research (SHINE-II). SPECIFIC AIM1 [A] Demonstrate aNILV-mediated phenotype correction, biosafety and clonal diversity under physiologic and pharmacological selection in a murine model of Fanconi Anemia (FA) associated bone marrow failure. SPECIFIC AIM1 [B] Track molecular persistence and multilineage expression during serial transplantation of aNILV transduced human CD34 progenitor cells in immunodeficient mice. SPECIFIC AIM1 [C] Design a minicircle-delivering lentivector (aNILV-II) and systematically determine its performance in clonally-derived cell lines and in vivo. The proposed platform promises to reconcile the efficiency of lentivector delivery with the inherent biosafety of episomally maintained genomes. This vector system will have a strong translational impact on stem cell gene therapy and may allow for significant cost savings, currently associated with long- term integration site surveillance in patients. !